Fred Snyder

Tetrahydropteridine-dependent cleavage enzyme for O-alkyl lipids: substrate specificity

The tetrahydropteridine-requiring enzyme in rat liver microsomes that oxidatively cleaved the O-alkyl moiety of glycerolipids does not utilize alkyl glycerolipid substrates that contain carbonyl, acyl, or phosphate groups on the glycerol portion. However, the ether linkage in rac and 1-, 2-, or 3-isomeric forms of alkylglycerols and 1-alkyl-sn-glycero-3-phosphorylethanolamine is readily cleaved by the pteridine-requiring enzyme. Therefore, it appears that hydrolytic enzymes play an essential role in conjunction with the cleavage enzyme in the regulation of cellular levels of O-alkyl moieties in glycerolipids.

Membrane lipid modification and stearoyl-coenzyme A desaturase activity in L-M cells.

Abstract The stearoyl-coenzyme A desaturase system of L-M cells, grown as monolayers, was examined in microsomal membranes that contained 8.2% phosphatidylisopropylethanolamine, an unnatural phospholipid analog. Desaturation of both [1- 14 C]stearic acid by whole cells and [1- 14 C]stearoyl-coenzyme A by cell-free homogenates, or microsomes, was decreased to about 40% of control levels in cells that had been grown for 24 h in the presence of 10 m m N -isopropylethanolamine. No decrease in microsomal NADH- or NADPH-dependent cytochrome c reductase activities or the level of cytochrome b 5 was found in the L-M cells that had been treated for 24 h with N -isopropylethanolamine. Although amino acid transport into L-M cells was not affected by treatment with N -isopropylethanolamine, protein synthesis was decreased by about 30%. These results indicate that the decrease in stearoyl-coenzyme A desaturation in the modified membranes is specifically associated with the terminal oxidase activity (cyanide-sensitive factor) of the desaturase enzyme complex.

The synthesis of14C- and3H-labeled glycerol ethers

The racemic14C-and3H-labeled alpha and beta derivatives of octadecyl glycerol ether (batyl alcohol) and of hexadecyl glycerol ether (chimyl alcohol) of high specific activity were synthesized by treating the appropriate alkyl halides with a large excess of the potassium salts of isopropylidene or benzylidene glycerol. By use of the trifluoroacetic anhydride esterification procedure, the labeled diesters of alpha and beta octadecyl and hexadecyl glycerol ethers were prepared. The labeled monoesters of beta octadecyl and of beta hexadecyl glycerol ethers were isolated from the reaction mixtures by silicic acid column chromatography.

Cardiovascular and Sympathetic Effects of 1-O-Hexadecyl-2-Acetyl-sn-Glycero-3-Phosphocholine in Conscious Shr and Wky Rats

Injections of 1-)-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (alkylacetyl-GPC, 0.2-5.0 nmol/300 g body weight) induced dose-related hypotension and tachycardia in spontaneous hypertensive (SHR) and normotensive control (WKY) rats. The hypotension that developed was more pronounced in SHR than in WKY rats and was unchanged by indomethacin pretreatment. Plasma norepinephrine (NE) and epinephrine (EPI) levels were markedly increased at the time of maximal hypotension (2 min after injection of alkylacetyl-GPC); plasma EPI (but not NE) was higher in the SHR than in WKY animals. Plasma levels of TXB2, but not 6-keto-PGF 1 alpha, increased in both groups; the increase was more pronounced in SHR than in WKY rats. In pithed SHR rats, alkylacetyl-GPC caused only short lasting hypotension without any effect on heart rate or circulating levels of NE or EPI. These data suggest that there is an increased vascular sensitivity to alkylacetyl-GPC in SHR rats and activation of thromboxane-generating elements in both SHR and WKY rats.

An alternate enzymic route for the synthesis of the alkyl analog of phosphatidic acid involving alkylglycerol

A key intermediate in the biosynthesis of complex ether lipids is 1-alkyl-2-acyl-sn-glycero-3-phosphate. This lipid is known to be formed by enzymic reduction of alkyldihydroxyacetone phosphate and subsequent acylation. We have now obtained evidence for an alternate pathway for its formation from hexadecylglycerol. The reaction is catalyzed by a mitochondrial supernatant fraction prepared from mouse preputial gland tumors; CoA, ATP, and Mg++ are required as cofactors.

Thyrotropin-releasing hormone blocks the hypotensive effects of platelet-activating factor in the unanesthetized guinea pig.

Platelet-activating factor (PAF) and leukotrienes, newly described classes of vasoactive lipids, may play a role in anaphylaxis. It has recently been suggested that the vasoconstrictor effects of PAF in isolated rat lung are related to release of leukotrienes C4 and D4. Thyrotropin-releasing hormone (TRH), a tripeptide, has potent antihypotensive activity in experimental shock, including that resulting from either leukotriene D4 administration or antigen-induced anaphylaxis. We utilized an unanesthetized guinea pig model to study the relationships among PAF, leukotrienes, and TRH and their potential interactions on the cardiovascular system. PAF (1 nmol/600 g body weight i.v.) produced profound hypotension which was completely blocked by TRH (2 mg/kg i.v.). Nafazatrom or FPL 55712, a presumed receptor antagonist of leukotrienes, was ineffective, whereas U-60257, a leukotriene synthesis inhibitor, displayed incomplete blockade. Moreover, leukotriene-like immunoreactivity in plasma did not increase following PAF administration. Thus, hypotension produced by PAF does not appear to result secondarily from release of cysteinyl leukotrienes. Moreover, the ability of TRH to block the hypotensive effects of PAF may partially account for its beneficial effects in experimental anaphylaxis and provides further rationale for the therapeutic evaluation of this peptide in anaphylactic shock.

Biosynthesis of alkyl lipids: Displacement of the acyl moiety of acyldihydroxyacetone phosphate with fatty alcohol analogs

Abstract The first step in the biosynthesis of ether-linked glycerolipids proceeds as follows: ROH + acyldihydroxyacetone-P → alkyldihydroxy-acetone-P + RCOOH. Data obtained with a series of 3 H-labeled fatty alcohol analogs and [1- 14 C]hexadecanol demonstrate that the microsomal enzyme from tumors that substitutes the alcohol group for the acyl group of acyl-dihydroxyacetone-P is not very selective. However, if hydroxyl groups are inserted at either the C-2 or C-16 position of hexadecanol, neither alkyl-dihydroxyacetone-P nor its dephosphorylated product is formed. The effect of modifying the terminal end of the alcohol was also apparent when iso and anteiso branched chain alcohols were used as substrates, i.e., the latter was incorporated into alkyldihydroxyacetone-P to a much greater extent.

Infrared spectra and polymorphism of glycerol ether derivatives

Abstract The infrared spectra and the polymorphic forms of the α- and β-glycerol ethers, glycerol ether monoesters, and glycerol ether diesters were investigated. It was concluded from these studies that the α-ethers exist in at least two different crystalline forms, whereas the β-ethers exist in only one form. The stable A form of both isomers of the ethers probably has hexagonal packing of the side chains; however, the B form of the α-ethers may have triclinic packed chains. It was found that the α-monoesters β-ethers and the β-monoester α-ethers also exist in at least two crystalline forms. The lower melting form B of the β-monoester α-ether does not correspond in crystalline structure to the lower melting form B of the α-monoester β-ether. The results indicate that there are at least three or possibly four crystalline forms of dipalmitoyl-α-batyl alcohol. The data imply that form D of dipalmitoyl-α-batyl alcohol is converted to form A and also that form B is converted to form A. It was found that the β-ether diesters are stable at room temperature in probably an orthorhombic arrangement, whereas the α-ether diesters are stable only in a triclinic or hexagonal arrangement. A comparison of the polymorphic forms of glycerides and glycerol ether derivatives demonstrated that both classes of compounds exist in a large number of similar crystalline forms; however, the glycerol ether derivatives may also exist in forms which have not yet been reported for the glycerides.

Enzymic studies of glycol and glycerol lipids containing O-alkyl bonds in liver and tumor tissues☆

Abstract The utilization of [1- 14 C]hexadecyl-[2- 3 H]ethyleneglycol and [1- 14 C]hexadecyl-[2- 3 H]glycerol as substrates for acyltransferase, phosphotransferase, phosphorylcholine, and phosphorylethanolamine transferase, and O -alkyl cleavage activities in cell-free preparations from normal rat liver and preputial gland tumors of mice was investigated. Our studies demonstrate that alkylethyleneglycols, like alkylglycerols, can serve as substrates for acyltransferases in both the liver and tumor microsomes; the product alkylacylethyleneglycerol can be readily deacylated by pancreatic lipase. A polar lipid was formed from the alkylethyleneglycol by the tumor homogenates in the presence of ATP and Mg 2+ ; although the small quantities formed precluded absolute identification, its thin-layer Chromatographic behavior in acidic and basic solvent systems indicated that a free phosphate group was present. As expected, phosphorylbase transferases in these preparations did not utilize either the alkylethyleneglycol or alkylglycerol as substrates. The O -alkyl moiety of hexadecyl-ethyleneglycol was oxidized to hexadecanal by a tetrahydropteridine-dependent cleavage enzyme in rat liver microsomes, whereas in the tumor microsomes this activity was not present. We conclude that alkylethyleneglycols are metabolized in a manner similar to alkylglycerols and perhaps by identical enzymes.

Labeling and radiopurity of lipids.

Publisher Summary This chapter presents a survey of the methods of labeling lipid molecules with radioactive elements by organic synthesis, biosynthesis, and isotope-exchange techniques. It also discusses procedures that can be employed for establishing the purity of lipid compounds. Chromatographic procedures are used to establish both radioactive and chemical purities of lipids for biological experiments. Thin-layer chromatography and gas–liquid chromatography complement each other very well when used to check lipid purity, as the former separates lipids into classes by adsorption processes and the latter can separate the resulting individual lipid classes into homologs by partition processes. In this way, for instance, fatty acids can be isolated from monoglycerides, diglycerides, and triglycerides by thin-layer chromatography; then, the constituent fatty acids of each of these classes can be defined by gas–liquid chromatography.